16S RNA Methylase Research Articles

Epidemiology and antibiotic resistance trends in clinical isolates of Pseudomonas aeruginosa from Rio de janeiro - Brazil: Importance of mutational mechanisms over the years (1995–2015)

Pseudomonas aeruginosa is a major health concern globally and treating infections caused by MDR-isolates unarguably a humongous challenge that remains an unmet need in modern medicine. To determine patterns and mechanisms of antimicrobial resistance and its spread over the years in Rio de Janeiro, Brazil, 88 P. aeruginosa isolates were selected from 1995 to 2015. Phenotypic and genotypic characterization of antimicrobial resistance was evaluated and isolates were submitted to clonality by PFGE and MLST. PFGE analysis showed a great variability of clonal groups mainly over the past 10 years of this study. STs predominant in the early years (ST804, ST1860, ST487 and ST1602) associated to multidrug resistance (MDR) phenotype were replaced by ST277, ST244, ST1945, ST1791 with extensive drug resistance (XDR) in last years, with significant increase in resistance to carbapenems, fluoroquinolones and aminoglycosides. Colistin resistance was detected in 3.5%. The main mechanisms of antimicrobial resistance were mutational mechanisms (mutations in oprD, mexT and gyrA genes). We found the ESBL genes blaTEM (n = 2), blaSHV (n = 3) and blaCTX (n = 1).The carbapenemases genes was present in ST277 (blaSPM, n = 3), ST1560 (blaKPC, n = 3) and ST1944 (blaKPC, n = 2). The 16S RNA methylase gene (rmtD) was found in five isolates belonged to ST277. In conclusion, molecular epidemiological investigation reveals an increase of antimicrobial resistance in P. aeruginosa over 21 years in Rio de Janeiro with higher population structure and occurrence of high risk clone in the last years. The mutational mechanisms of resistance were present in all XDR isolates.

First Occurrence of OXA-72-Producing Acinetobacter baumannii in Serbia.

Here, we characterized the first OXA-72-producing Acinetobacter baumannii isolate (designated MAL) recovered from a urine sample from a Serbian patient. Antimicrobial susceptibility testing, plasmid analysis, and whole-genome sequencing (WGS) were performed to fully characterize the resistome of the A. baumannii MAL clinical isolate. The isolate was multidrug resistant and remained susceptible only to colistin and tigecycline. PCR analysis revealed the presence of the carbapenemase OXA-72, an OXA-40 variant. Extraction by the Kieser method revealed the presence of two plasmids, and one of these, a ca. 10-kb plasmid, harbored the blaOXA-72 gene. WGS revealed 206 contigs corresponding to a genome of 3.9 Mbp in size with a G+C content of 38.8%. The isolate belonged to sequence type 492 and to worldwide clone II (WWCII). Naturally occurring β-lactamase-encoding genes (blaADC-25 and blaOXA-66) were also identified. Aminoglycoside resistance genes encoding one aminoglycoside adenyltransferase (aadA2), three aminoglycoside phosphatases (strA, strB, aphA6), and one 16S RNA methylase (armA) conferring resistance to all aminoglycosides were identified. Resistance to fluoroquinolones was likely due to mutations in gyrA, parC, and parE Of note, the resistome matched perfectly with the antibiotic susceptibility testing results.

First report in China of Enterobacteriaceae clinical isolates coharboring blaNDM-1 and blaIMP-4 drug resistance genes.

To describe the identification of two carbapenem-resistant, NDM-1 and IMP-4, carbapenemases coproducing Enterobacteriaceae isolates recovered from hospitalized patients in China. Both Klebsiella pneumoniae clinical isolates (Kpn922 and Kpn9599) were resistant to meropenem and imipenem and were subjected to additional antibiotic susceptibility testing. Polymerase chain reaction (PCR) and sequence analyses were used to characterize bacterial carbapenemase resistance genes, extended-spectrum β-lactamases, plasmid-mediated AmpC enzymes, quinolone resistance, and 16s RNA methylase. Genetic relatedness was determined using pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). Plasmids were analyzed by S1-PFGE and Southern blot. PCR analyses revealed that the Kpn922 isolate carried blaNDM-1, blaIMP-4, blaTEM-1, and blaSHV-1 genes, while Kpn9599 carried blaNDM-1, blaIMP-4, blaTEM-1, and blaSHV-12 genes. MLST determined that the two isolates were ST1043 and ST571 sequence types. Southern blot analyses revealed that metallo-β-lactamase genes were plasmid borne in both isolates. Plasmids ∼300 kb simultaneously carried blaNDM-1 and blaIMP-4. Coexistence of blaNDM-1 and blaIMP-4 in these clinical isolates may herald the emergence of a new pattern of drug resistance. Surveillance of carbapenemases, particularly metallo-β-lactamases, in Enterobacteriaceae is urgently needed to control and prevent the spread of these resistance determinants in China.

Complete sequencing of an IncHI1 plasmid encoding the carbapenemase NDM-1, the ArmA 16S RNA methylase and a resistance-nodulation-cell division/multidrug efflux pump

To characterize the pNDM-CIT plasmid identified in Citrobacter freundii carrying genes encoding the metallo-β-lactamase NDM-1 and the 16S RNA methylase ArmA. The complete DNA sequence of pNDM-CIT was obtained by using the 454-Genome Sequencer FLX procedure on a library obtained using plasmid DNA purified from the pNDM-CIT Escherichia coli J53 transconjugant. Contig assembly and predicted gaps were confirmed and filled by PCR-based gap closure. Comparative analysis with IncHI1 incompatibility group plasmids was performed using BLASTN and BLASTP algorithms. Plasmid pNDM-CIT was 288::920 bp and revealed an IncHI1 plasmid scaffold, showing novel resistance and potential virulence determinants. The bla(NDM-1) gene was identified within a novel genetic context, flanked by a duplication of the class 1 integron on both sides. The replicase gene repAciN, originating from Acinetobacter spp. plasmids, was identified in a close association with the Tn1548::armA transposon and the macrolide resistance mel-mph2 cluster. The same structure was identified in silico from a series of enterobacterial plasmids carrying the armA gene. The repAciN gene probably represents a remnant sign of the original occurrence of the armA gene in Acinetobacter plasmids. A CP4-like prophage sequence was identified in pNDM-CIT, containing a resistance-nodulation-cell division/multidrug resistance (RND/MDR) efflux pump cluster surrounded by two IS1-like elements. This resistance determinant, associated with such a prophage sequence, has never been reported on plasmids. Plasmid pNDM-CIT differed significantly from all known bla(NDM-1)-carrying plasmids identified in Enterobacteriaceae, since it combines the metallo-β-lactamase NDM-1, the 16S RNA methylase ArmA and a cryptic prophage carrying the RND/MDR efflux pump.

Emergence of Klebsiella pneumoniae isolate co-producing NDM-1 with KPC-2 from India

Sir, The emergence of NDM-1-producing isolates and their sources have been clearly identified in several countries worldwide. In particular, the blaNDM-1 gene was identified in various genera of Enterobacteriaceae and in non-fermenting Gram-negative bacilli from environmental samples in India. Furthermore, the increasing co-production of NDM-1 with other carbapenemases has been detected amongst isolates of Enterobacteriaceae and Acinetobacter sp. in many parts of India. – 7 We report here the isolation of a strain of Klebsiella pneumoniae (designated IR98) from a urine sample from a middle-aged patient admitted to the intensive care unit of a tertiary care hospital in Chennai, India in July 2010. Species identification and antibiotic susceptibility, determined using an automated system (VITEK-2, bioMerieux Inc.), showed wide-spectrum resistance to b-lactams, aminoglycoside, fluoroquinolones, co-trimoxazole, nitrofurantoin and tigecycline, but susceptibility to colistin and fosfomycin, according to CLSI guidelines. MICs of various antibiotics were determined using the agar dilution method, while the tigecycline MIC was determined using the broth microdilution method. European Committee on Antimicrobial Susceptibility Testing (EUCAST) breakpoints were applied to interpret the susceptibilities. The isolate was highly resistant to imipenem (256 mg/L), meropenem (128 mg/L), ceftazidime (.256 mg/L), cefotaxime (.256 mg/L), amikacin (.512 mg/L), gentamicin (.512 mg/L), tobramycin (.512 mg/L), netilmicin (.512 mg/L), co-trimoxazole (.32 mg/L), ciprofloxacin (.32 mg/L) and tigecycline (4 mg/L), but remained susceptible to colistin (0.5 mg/L). The double-disc synergy test (DDST), modified Hodge test (MHT) and combined-disc synergy test (CDST) were used for the detection of metallo-b-lactamases (MBLs), other carbapenemases and KPC, or KPC with MBLs. The simultaneous production of MBL and KPC-like carbapenemases was confirmed by positive DDST, MHT and CDST with meropenem discs containing both EDTA and phenylboronic acid (PBA) or EDTA, while meropenem discs supplemented with PBA were negative. PCR assays for genes encoding b-lactamases and 16S rRNA methylases revealed the presence of blaNDM-1, blaKPC-2, blaCTX-M-15, blaSHV-12, blaTEM-1, blaOXA-1 and rmtB genes. Plasmid analysis using the Kieser technique revealed that K. pneumoniae IR98 harboured four plasmids, with sizes of 160, 120, 70 and 40 kb, using Escherichia coli NCTC 50192 as a reference. To study the transferability of these plasmids (encoding the resistance determinants), transconjugation and transformation experiments were performed using E. coli J53 (Azide-R) and E. coli TOP10 as recipient strains. Transconjugants were selected on MacConkey agar plates using sodium azide (200 mg/L) with ceftazidime (2 mg/L), meropenem (0.5 mg/L) or amikacin (20 mg/L). The plasmid extract of K. pneumoniae IR98 was transformed into E. coli TOP10, and transformants were selected on MacConkey agar plates containing 2 mg/L ceftazidime. Selected colonies were replica-plated onto MacConkey agar plates with or without meropenem (0.5 mg/L) or amikacin (20 mg/L). The genes encoding NDM-1, CTX-M15 and 16S rRNA methylase were transferred in conjugation experiments, whereas transfer of KPC-2 was successful only by transformation. The plasmids purified from the clinical isolate, transconjugants and transformants were typed by PCR-based replicon typing (PBRT). The E. coli J53-p98A transconjugant obtained from the meropenem plate showed an MBL phenotype and elevated MICs of all the b-lactams (except aztreonam) and susceptibility to non-b-lactam antibiotics. Plasmid analysis revealed that E. coli J53-p98A harboured a 160 kb plasmid that belonged to the Inc A/C type. Subsequently, this plasmid was found by PCR to carry the blaNDM-1 gene. In addition, the E. coli J53-p98B transconjugant grown on both ceftazidime and amikacin plates showed decreased susceptibility to aminoglycosides and cephalosporins except cefoxitin, and was positive for extendedspectrum b-lactamase (ESBL) production on DDST. Both blaCTX-M15 and rmtB genes were carried on a 120 kb IncF plasmid that was identified in E. coli J53-p98B. In contrast, the ESBL-negative transformant (E. coli TOP10-p98C) from both ceftazidime and meropenem plates was resistant to all the b-lactams except carbapenems (MICs 2 mg/L). The blaKPC-2 gene together with blaTEM-1 was carried on a 70 kb non-typeable plasmid that was identified in E. coli TOP10-p98C. Although the co-existence of blaNDM-1 with different carbapenemase genes has been reported in India, we believe this to be the first report of the co-occurrence of blaNDM-1 with blaKPC-2 and rmtB in a clinical isolate of K. pneumoniae from India. This co-production of NDM-1 with an unrelated carbapenemase and 16S RNA methylase results in very broad-spectrum antibiotic resistance profiles. The growing emergence of these powerful resistance mechanisms in India is cause for great concern as treatment options are virtually exhausted.

Genetic Features of bla NDM-1 -Positive Enterobacteriaceae

Genetic features associated with the bla(NDM-1) gene were investigated in 6 Escherichia coli, 7 Klebsiella pneumoniae, 1 Citrobacter freundii, 1 Proteus mirabilis, and 1 Providencia stuartii isolate of worldwide origin. Clonal diversity was observed for both E. coli and K. pneumoniae. The bla(NDM-1) gene was carried by different plasmid types (IncA/C, IncF, IncL/M, or untypeable) and was likely chromosome borne in two isolates. The bla(NDM-1) plasmids coharbored a variety of resistance determinants, including β-lactamase genes, quinolone resistance genes, and 16S RNA methylase genes.

Analysis of the Resistome of a Multidrug-Resistant NDM-1-Producing Escherichia coli Strain by High-Throughput Genome Sequencing

The resistome of the multidrug-resistant Escherichia coli strain 271 carrying the plasmid-mediated bla(NDM-1) carbapenemase gene was analyzed by high-throughput genome sequencing. The p271A plasmid carrying the bla(NDM-1) gene was 35.9 kb in size and possessed an IncN-type backbone that harbored a novel replicase gene. Acquisition of the bla(NDM-1) gene on plasmid p271A had been likely the result of a cointegration event involving the transposase of Tn5403. The expression of bla(NDM-1) was associated with the insertion sequence ISAba125 likely originating from Acinetobacter baumannii. E. coli 271 accumulated multiple resistance determinants, including five β-lactamase genes (comprising the extended-spectrum β-lactamase CTX-M-15), two 16S RNA methylase ArmA- and RmtB-encoding genes, and the qepA gene encoding an efflux pump involved in resistance to fluoroquinolones. These resistance genes were located on three additional plasmids, of 160 kb (IncA/C), 130 kb (IncF), and 110 kb (IncI1). In addition, several chromosomally encoded resistance determinants were identified, such as topoisomerase mutations, porin modifications and truncations, and the intrinsic ampC gene of E. coli that was weakly expressed. The multidrug resistance pattern observed for E. coli 271 was therefore the result of combined chromosome- and plasmid-encoded mechanisms.

Outbreak of Salmonella enterica serotype Infantis producing ArmA 16S RNA methylase and CTX-M-15 extended-spectrum β-lactamase in a neonatology ward in Constantine, Algeria

Plasmid-mediated 16S rRNA methylases such as ArmA, which confer high levels of resistance to aminoglycosides, are increasingly reported in Enterobacteriaceae. This study investigated the molecular mechanism of β-lactam and aminoglycoside resistance in extended-spectrum β-lactamase (ESBL)-producing Salmonella enterica serotype Infantis isolated at the 53-bed neonatology ward of University Hospital Benabib in Constantine, Algeria. From September 2008 to January 2009, 200 S. enterica isolates were obtained from 138 patients (age range 8–80 months) hospitalised in the neonatology ward. Most isolates were from stool cultures, but also from two blood cultures and one gastric fluid. The isolates were multidrug-resistant and produced TEM-1 and CTX-M-15 enzymes as well as the 16S RNA methylase ArmA. The armA, bla CTX-M-15 and bla TEM-1 genes were located on the same 140-kb self-transferable plasmid belonging to the IncL/M incompatibility group. All of the S. Infantis isolates belonged to a single clone. Increased infection control measures and thorough biodecontamination of the rooms led to control of the outbreak but did not eradicate the epidemic strain. This study further illustrates the global emergence of ArmA methylase and its frequent association with bla CTX-M genes. Spread of 16S RNA methylase determinants at the same level as bla CTX-M genes in Enterobacteriaceae may seriously compromise the efficacy of aminoglycosides for treating Gram-negative infections.

NDM-2 carbapenemase in Acinetobacter baumannii from Egypt

To analyse the mechanisms responsible for carbapenem resistance in one Acinetobacter baumannii isolate recovered from a patient transferred to Germany from an Egyptian hospital. PCR and sequencing were used to search for β-lactamase and 16S RNA methylase genes. Multilocus sequence typing was used to determine the sequence type (ST) of the isolate. Sequencing of the PCR product obtained using primers for bla(NDM-1) revealed a variant of NDM-1 that had a C to G substitution at position 82 resulting in an amino acid substitution of proline to alanine at position 28. This variant was designated NDM-2. Genes encoding extended-spectrum β-lactamases or 16S RNA methylase were not detected. The strain lacked detectable plasmids and bla(NDM-2) was not transferred by conjugation. MLST showed that the isolate belonged to a new ST, ST103. This work further underlines the spread of NDM carbapenemases in A. baumannii, and the spread of the corresponding gene in the Middle East. It also describes the first variant of NDM-1.

PER-7, an Extended-Spectrum β-Lactamase with Increased Activity toward Broad-Spectrum Cephalosporins in Acinetobacter baumannii

Acinetobacter baumannii isolate AP2 was recovered from a bronchial lavage sample of a patient hospitalized in Paris, France. A. baumannii AP2 was resistant to all β-lactams, including carbapenems, and expressed the extended-spectrum β-lactamase (ESBL) PER-7, which differs from PER-1 by 4 amino acid substitutions. Compared to PER-1, PER-7 possessed higher-level hydrolytic activities against cephalosporins and aztreonam. The blaPER-7 gene was chromosomally located and associated with a mosaic class 1 integron structure. Additionally, isolate AP2 expressed the carbapenem-hydrolyzing oxacillinase OXA-23 and the 16S RNA methylase ArmA, conferring high-level resistance to aminoglycosides.

International Transfer of NDM-1-Producing Klebsiella pneumoniae from Iraq to France

The carbapenemase NDM-1 was initially identified in Escherichia and Klebsiella pneumoniae in Sweden from a patient transferred from India (10). Since then, it has been identified in many enterobacterial species and isolates from patients in the United Kingdom, India, and Pakistan (4). In addition, recent reports have indicated the spread of NDM-1 producers in many different countries, including Austria, Australia, Belgium, Canada, Denmark, France, Germany, Kenya, the Netherlands, Norway, the Sultanate of Oman, and the United States (6-9). Most of these reports indicated a likely source of NDM-1 producers located in the Indian subcontinent, with both hospital and community acquisition. However, a detailed analysis of the clinical cases indicated that importation of NDM-1 isolates may also have originated from Balkan countries, such as Kosovo, Serbia, Montenegro, and Bosnia and Herzegovina, which may constitute an additional reservoir for NDM-1 producers (6). A 22-year-old Iraqi male resident in Baghdad was admitted at the Bicetre Hospital (suburb of Paris) on 8 November 2010, following a direct transfer from Baghdad, Iraq. He had been a victim of a terrorist attack at the Baghdad cathedral that killed over 50 people on 31 October 2010. He had a wounded shoulder and was immediately hospitalized at the Ibn Al Nafees Hospital of Baghdad, where he was operated on and stayed for 5 days. At the Bicetre Hospital, he was screened for multidrug-resistant bacteria, including extended-spectrum β-lactamase (ESBL) and carbapenemase producers, methicillin-resistant Staphylococcus aureus, and glycopeptide-resistant enterococci. Rectal swabs grew only on ChromID ESBL culture medium (bioMerieux, La Balme-les-Grottes, France) (5). K. pneumoniae isolate IBN was obtained, which showed resistance or decreased susceptibility to all β-lactams, including carbapenems (the MICs of imipenem, ertapenem, doripenem, and meropenem were 2, 8, 2, and 3 μg/ml, respectively), according to the CLSI's updated guidelines (1). It was also resistant to gentamicin, kanamycin, tobramycin, sulfonamides, rifampin, chloramphenicol, and fluoroquinolones but remained susceptible to amikacin and fosfomycin, the MICs of tigecycline and colistin being 0.25 and 0.5 μg/ml, respectively. The patient did not develop any infection while hospitalized in France. He was neither treated with antibiotics nor decontaminated (actually not recommended for carriage of multidrug-resistant Enterobacteriaceae). PCR, sequencing, and plasmid analysis revealed that K. pneumoniae IBN harbored the blaNDM-1 carbapenemase gene, in addition to a blaCTX-M-15 ESBL gene, each located on a different plasmid (100 and 160 kb in size, respectively). Screening for additional β-lactamase genes and for 16S RNA methylase genes, as reported previously (9), showed that K. pneumoniae IBN was coharboring the blaSHV-11 and blaOXA-1 genes, but no 16S RNA methylase gene was identified. Multilocus sequence typing was performed as described worldwide (3) to evidence a possible link with other recently identified NDM-1-producing K. pneumoniae isolates, and the results were analyzed by eBURST (http://pubmlst.org). The results showed that isolate IBN belonged to the ST147 sequence type, whereas the NDM-1-positive K. pneumoniae 05-506 index strain and K. pneumoniae isolates recovered in India and in the Sultanate of Oman belonged to the ST14 type, both types differing significantly (2, 7). Interestingly, the only report of ST147-type K. pneumoniae so far corresponds to a clonal spread that has been identified in Hungary (2). This Hungarian ST147 clone was susceptible to carbapenems, but it harbored a blaCTX-M-15 plasmid and was resistant to fluoroquinolones (2), as observed for isolate IBN from Iraq. It may therefore be speculated that these isolates could be clonally related. Considering that one of the two NDM-1-producing K. pneumoniae isolates previously identified from Oman could not be traced back to the Indian subcontinent, this report provides an additional clue that the Middle East might also be a reservoir for NDM-1 producers. This result should be taken in account when taking care of any civilian or soldier hospitalized in Iraq and transferred abroad. It also suggests that the spread of NDM-1 producers is already much wider than suspected. Finally, it strengthens the value of systematic screening for multidrug-resistant bacteria for preventing the development of nosocomial outbreaks of carbapenem-resistant Enterobacteriaceae, as recommended in reference 6.

NDM-1-producing Klebsiella pneumoniae isolated in the Sultanate of Oman

To analyse the mechanisms responsible for multidrug resistance in two carbapenem-resistant Klebsiella pneumoniae isolates recovered from patients hospitalized in Oman. PCR and sequencing were used to search for β-lactamase and 16S RNA methylase genes. Multilocus sequence typing was used to determine the sequence type (ST) of each isolate. Clonal relationships were evaluated by PFGE. Both isolates carried the bla(NDM-1) carbapenemase gene. Isolate 601 was recovered from a patient who was transferred from India, whereas isolate 419 was from an Omani patient who had not travelled abroad. The two isolates were clonally unrelated, and belonged to ST14 (isolate 601) and ST340 (isolate 419). In addition to NDM-1, the ST14 isolate expressed β-lactamases CTX-M-15, SHV-28, OXA-1, OXA-9 and TEM-1, and the aminoglycoside resistance methylase ArmA. The ST340 isolate expressed β-lactamases SHV-11, OXA-1 and ArmA. In both isolates, the bla(NDM-1) gene was located on plasmids that were of similar size (170 kb), but of different incompatibility groups. This is the first description of NDM-1 producers in the Arabian peninsula and in the Middle East.

RmtD 16S RNA Methylase in Epidemiologically Unrelated SPM-1-Producing Pseudomonas aeruginosa Isolates from Brazil

We read with interest the article by Doi et al. (2) suggesting that the coproduction of the metallo-β-lactamase SPM-1 and the RmtD 16S RNA methylase is highly prevalent among imipenem-resistant Pseudomonas aeruginosa isolates from Brazil. SPM-1 is widespread among Brazilian carbapenem-resistant P. aeruginosa isolates, and isolates carrying blaSPM-1 have been observed in distinct regions of this country (3). The majority of SPM-1-producing isolates belong to a unique molecular clone identified by pulsed-field gel electrophoresis and automated ribotyping (3). The findings of Doi et al. (2) led us to test a very broad collection of epidemiologically unrelated blaSPM-1-carrying P. aeruginosa strains recovered from diverse Brazilian regions for the presence of rmtD. We evaluated 26 epidemiologically unrelated SPM-1-producing P. aeruginosa clinical isolates collected from different patients during 2002 in 10 Brazilian cities located in nine states (Table ​(Table1).1). These isolates belonged to 16 different ribogroups, and six isolates belonged to the major ribotype spread throughout the country (ribotype 72-3). Screening for rmtD was performed by PCR using the primers rmtD-F (5′-GAG CGA ACT GAA GGA AAA AC-3′) and rmtD-R (5′-CAG CAC GTA AAA CAG CTC-3′), which produce a 730-bp product. Amplicons were sequenced on both strands by using the ABIPrism 377 system (Applied Biosystems, Foster City, CA). The nucleotide sequences were analyzed using the Lasergene software package (DNASTAR, Madison, WI), and the sequences obtained were compared to sequences available over the Internet (http://www.ncbi.nlm.nih.gov/blast/Blast.cgi). TABLE 1. Characteristics of the 26 epidemiologically unrelated SPM-1-producing P. aeruginosa clinical isolates from different regions in Brazil Among the 26 isolates, rmtD was detected in only 4 (15.4%) strains. This rate is much lower than the results obtained previously (51.0%), implying that the production of RmtD is not prevalent among SPM-1-producing isolates with distinct genetic backgrounds and/or from different geographic areas in Brazil. blaSPM-1 is different from other metallo-β-lactamase genes that are carried in class 1 integrons also harboring aminoglycoside resistance genes as part of other gene cassettes (5) in that blaSPM-1 is embedded in a distinct genetic element (4) where the correlation of β-lactam and aminoglycoside resistance is not as evident. However, many SPM-1-producing isolates are resistant to other antimicrobial agents, including aminoglycosides. Recently, Carvalho et al. (1) reported that all 13 SPM-1-producing P. aeruginosa isolates found in another Brazilian state carried a class 1 integron, named In163, harboring two aminoglycoside resistance genes, aacA4 and aadA7, showing that aminoglycoside resistance among SPM-1-producing isolates can be also encoded by distinct resistance mechanisms other than the RmtD 16S RNA methylase gene.